The present invention relates to cellobiose dehydrogenase (CDH) enzymes, modifications thereof and electrochemical uses.
Cellobiose dehydrogenase (EC 1.1.99.18, CDH) was first discovered 1974 in the extracellular enzyme system of Phanerochaete chrysosporium and later on in several other basidiomycete fungi. A special characteristic of this enzyme is its composition: the combination of a catalytically active flavin domain, hosting a non-covalently bound FAD, and a haem domain, with a haem b as a cofactor. Both domains are connected by a flexible linker. By its catalytic activity the natural substrate cellobiose is oxidised in a reaction which reduces the FAD of the flavin domain. Subsequently, FAD can be reoxidised by the action of the haem domain. The spectral characteristics of a typical CDH clearly show the presence of both cofactors. Another characteristic described is the strong glucose discrimination of all well characterised enzymes. Until the discovery of the ascomycete fungus Myriococcum thermophilum (Stoica et al., 2005, Biosensors and Bioelectronics 20: 2010-2018; Harreither et al., 2007, Electroanalysis 19: 172-180), CDH was believed to strongly inhibit the conversion of glucose (Henriksson et al., 1998, Biochimica Biophysica Acta 1383: 48-54). Similarly, for a long time only CDHs exhibiting an acidic activity optimum were known, especially when the haem domain is involved in catalysis as it depends on intramolecular electron transfer (IET), which is necessary to transfer electrons via the haem to the electron acceptor. This is the case with cytochrome c in enzymatic assays, as well as on electrode surfaces where the haem domain enables direct electron transfer (DET) to the electrode.
Electrochemical applications described in the literature are the detection of cellobiose, cello-oligosaccharides, lactose and maltose soluble cellodextrins, ortho- and para-diphenolic compounds (Lindgren et al., 1999, Analyst 124: 527-532) and catecholamines (Stoica et al., 2004, Analytical Chemistry, 76: 4690-4696) mostly by mediated electron transfer (MET). So far the application in glucose biosensors based on the direct electron transfer (DET) properties of CDH was prevented by i) a very low or no glucose turnover, ii) the acidic pH optimum of most known CDHs and iii) a bad performance of some CDHs on electrodes.
Although, one CDH with well functioning IET at neutral or alkaline pH values is known (from the fungus Humicola insolens), it was shown not to convert glucose (Schou et al., 1998, Biochemical Journal 330: 565-571). One CDH currently known to convert glucose with significant turnover numbers was found in cultures of Myriococcum thermophilum (Harreither et al., 2007, Electroanalysis 19: 172-180). However, this enzyme has an acidic pH optimum for the IET and shows no activity under physiological pH conditions (pH 7.4). Another obstruction is the sometimes bad electronic communication of a CDH with an electrode surface, like the Humicola insolens and Sclerotium rolfsii CDHs (Lindgren et al., 2001, Journal of Electroanalytical Chemistry 496: 76-81), which results in very low current densities and therefore low signals even with the natural substrate cellobiose.
Harreither et al. (Electroanalysis, 19 (2-3) (2007): 172-180) disclose CDH direct electron transfer activity assays measured on an electrode. Activity at different pH values was determined with lactose or cellobiose as substrates. Although, glucose is accepted as a substrate at the pH optimum no information of the CDH activity on glucose at pH 7.4 is given. As is shown in the comparative examples herein, the activity of wild type CDH of M. thermophilum steeply decreases at neutral pH values above pH 5.5 and has no activity on glucose at pH 7.4.
Zamocky et al. (Prot. Expr. Pur. Acad. Press; 59 (2) (2007): 258-265) discloses the wild type M. thermophilum CDH and its DCIP activity when using citrate as substrate. The DCIP activity does not relate to the IEP activity of the catalysis of carbohydrate oxidation reactions on an electrode.
Database UniProt, Acc. No. A9XK88 discloses the wild type CDH sequence of M. thermophilum. 
U.S. Pat. No. 6,033,891 A discloses a CDH of Humicola insolens which does not have a glucose oxidating activity.
Database EMBL, Acc. No. AF074951, AAY82220 and AAZ95701 provide sequences of the CDH from Thielavia heterothallica. This enzyme does not have an activity on glucose at pH 7.4.
Zamocky et al. (Current protein and peptide science, 7 (3) (2006); 255-280) provide a review of CDHs of basidomycetes and ascomycetes.